Processing of the matricellular protein hevin in mouse brain is dependent on ADAMTS4.

The matricellular SPARC family member hevin (SPARC-like 1/SPARCL-1/SC1/Mast9) contributes to neural development and alters tumor progression in a range of mammalian models. The distribution of hevin in mouse tissues was reexamined with a novel monoclonal antibody that discriminates between hevin and its ortholog SPARC. We now report proteolysis of hevin in many tissues, with the most extensive processing in the brain. We demonstrate a cleavage site within the hevin sequence for the neural tissue proteinase ADAMTS4. Digestion of hevin by ADAMTS4 in vitro produced fragments similar to those present in brain lysates. Monoclonal antibodies revealed a SPARC-like fragment generated from hevin that was co-localized with ADAMTS4 in vivo. We show that proteolysis of hevin by ADAMTS4 in the mouse cerebellum is important for the normal development of this tissue. In conclusion, we have identified the fragmentation of hevin by ADAMTS4 in the mouse brain and propose that this specific proteolysis is integral to cell morphology and extracellular matrix deposition in the developing brain.

The copper binding domain of SPARC mediates cell survival in vitro via interaction with integrin beta1 and activation of integrin-linked kinase.

Secreted protein acidic and rich in cysteine (SPARC) is important for the normal growth and maintenance of the murine lens. SPARC-null animals develop cataracts associated with a derangement of the lens capsule basement membrane and alterations in lens fiber morphology. Cellular stress and disregulation of apoptotic pathways within lens epithelial cells (LEC) are linked to cataract formation. To identify molecular targets of SPARC that are linked to this disorder, we stressed wild-type (WT) and SPARC-null LEC by serum deprivation or exposure to tunicamycin. SPARC enhanced signaling by integrin-linked kinase (ILK), a serine/threonine kinase known to enhance cell survival in vitro. In response to stress, an ILK-dependent decrease in apoptosis was observed in WT relative to SPARCg-null LEC. Co-immunoprecipitation and cross-linking of cell lysates revealed enhanced levels of a SPARC-integrin beta1 complex during stress. Competition with monoclonal antibodies and peptides indicated that the copper binding domain of SPARC is required for SPARC-mediated response to stress. Inhibiting the binding and/or activity of ILK, integrin beta1, or SPARC resulted in increased apoptosis of stressed LEC. We conclude that SPARC protects cells from stress-induced apoptosis in vitro via an interaction with integrin beta1 heterodimers that enhances ILK activation and pro-survival activity.

Expression of integrin-linked kinase in the murine lens is consistent with its role in epithelial-mesenchymal transition of lens epithelial cells in vitro.

PURPOSE: To evaluate the expression and location of integrin-linked kinase (ILK) within the mouse lens and to characterize the role of this protein during mouse lens epithelial cells (LEC) differentiation in vitro. METHODS: Transcription levels of ILK mRNA were determined by RT-PCR in cultured cells and lens tissue. ILK protein was detected by immunoblotting, immunocytochemistry, immunohistochemistry, and immunoprecipitation. A role for ILK in the outgrowth of LEC from dissected mouse lens explants was determined by the use of ILK short interfering RNA (siRNA). Affinity-purified polyclonal anti-recombinant human ILK IgG was prepared and characterized for these experiments. A comparison of several anti-ILK antibodies was performed by immunoblotting, immunoprecipitation, and ELISA. RESULTS: ILK was transcribed in LEC and lens fiber cells in vivo. ILK protein was expressed in the differentiating LEC at the equatorial region of the lens and, to a lesser extent, within the cortical and nuclear fiber cells. LEC in vitro produced copious ILK, which exhibited a filamentous pattern throughout the cytoplasm. The expression of ILK was increased during epithelial-mesenchymal-transition (EMT) of LEC from lens explants, whereas inhibition of ILK by siRNA delayed expression of the EMT markers smooth muscle alpha-actin and fibronectin. CONCLUSIONS: Analysis of ILK expression, localization, and activity in the mouse lens and cultured LEC is substantially facilitated by the generation of a multi-functional, polyclonal, affinity-purified anti-ILK antibody. Expressed in most tissues and cells lines, ILK is unexpectedly restricted to the equatorial LEC and differentiated fiber cells of the mouse lens. The occurrence of ILK expression with LEC differentiation is consistent with the positive regulatory function of ILK, which is revealed in a model of EMT in vitro. This is the first study to show the expression of ILK in the lens and its unique distribution pattern within cultured lens epithelia.

Absence of SPARC in lens epithelial cells results in altered adhesion and extracellular matrix production in vitro.

The matricellular protein SPARC (also known as osteonectin and BM-40) is expressed abundantly in lens epithelium. That SPARC-null mice exhibit early cataractogenesis, indicates a role for SPARC in the maintenance of lens transparency. Comparison of cultured wild-type and SPARC-null lens epithelial cells revealed significant changes in adhesion to different substrates. SPARC-null lens cells displayed enhanced attachment and spreading, focal adhesion formation, and resistance to trypsin detachment in comparison to wild-type cells. In the absence of SPARC, there was increased deposition of the ECM protein laminin-1 (LN-1). Proteins associated with focal adhesions were increased in SPARC-null versus wild-type lens cells: levels of alpha6-integrin heterodimers, talin, and paxillin phosphorylated on tyrosine were enhanced significantly, as was the association of beta1-integrin with talin and paxillin. Restoration of the wild-type phenotype in SPARC-null cultures was accomplished through genetic rescue by stable transfection of SPARC cDNA. Our findings indicate that SPARC is counter-adhesive for murine lens epithelial cells and demonstrate that multiple factors contribute to this activity. We also identify SPARC as a modulator of LN-1 secretion and deposition by these cells, an activity important in epithelial cell-ECM interactions in the ocular lens.